Recombinant Escherichia coli BL21 with LngA Variants from ETEC E9034A Promotes Adherence to HT-29 Cells

The CS21 pilus produced by enterotoxigenic Escherichia coli (ETEC) is involved in adherence to HT-29 intestinal cells. The CS21 pilus assembles proteins encoded by 14 genes clustered into the lng operon. Aim. This study aimed to determine whether E. coli BL21 (ECBL) transformed with the lng operon lacking the lngA gene (pE9034AΔlngA) and complemented in trans with lngA variants of ETEC clinical strains, as well as point substitutions, exhibited modified adherence to HT-29 cells. Methods. A kanamycin cassette was used to replace the lngA gene in the lng operon of the E9034A strain, and the construct was transformed into the ECBL strain. The pJET1.2 vector carrying lngA genes with allelic variants was transformed into ECBLpE9034AΔlngA (ECBLΔlngA). The point substitutions were performed in the pJETlngAFMU073332 vector. Results. Bioinformatic alignment analysis of the LngA proteins showed hypervariable regions and clustered the clinical ETEC strains into three groups. Variations in amino acid residues affect the adherence percentages of recombinant ECBL strains with lngA variants and site-specific mutations with HT-29 cells. Conclusion. In this study, ECBL carrying the lng operon harboring lngA variants of six clinical ETEC strains, as well as point substitutions, exerted an effect on the adherence of ECBL to HT-29 cells, thereby confirming the importance of the CS21 pilus in adherence.


Introduction
Enterotoxigenic Escherichia coli (ETEC) is responsible for nearly 220 million cases of diarrhea annually, and approximately 75 million occur in children under five years of age with 18,700 deaths according to the "Institute for Health Metrics and Evaluation" estimates and 42,000 deaths according to "Maternal-Infant Epidemiology" estimates [1,2]. ETEC-induced intestinal infections in children less than 5 years of age are particularly common among low-income populations and in areas with poor sanitary conditions [3,4]. ETEC is responsible for many cases of traveler's diarrhea worldwide, and the clinical symptoms include watery diarrhea induced by secretion of a thermolabile toxin (LT) and/or a thermostable toxin (ST) [5,6]. STa and LT-I are associated with the disease in both humans and animals, STb is mainly associated with diarrhea in piglets, and LT-II has only been associated with animal disease [7].

Cloning of the lngA Variants into the pJET1.2 Vector
DNA of the clinical ETEC strains was obtained from 200 µL of a bacterial suspension grown in LB broth to an OD 600 of 1.0. Briefly, the bacteria were resuspended in distilled water, boiled for 5 min, and centrifuged for 5 min at 25 g to obtain the total DNA. The lngA variants were amplified by PCR using Platinum R Taq DNA Polymerase High Fidelity (Invitrogen, Carlsbad, CA, USA) and forward-and reverse-specific primers ( Table 3). The lngA variants were cloned into the pJET1.2 vector following the instructions provided by the manufacturer (Thermo Fisher Scientific; Carlsbad, CA, USA) ( Table 4). The DNA variant product cloned into the pJET1.2 vector was sequenced using forward and reverse primers at the Institute of Genomic Services "LANGEBIO-CINVESTAV" Campus Irapuato, Mexico.

Transformation of ECBL with the lng Operon and lngA Variants
The 14-kb plasmid harboring the lng operon of the CS21 pilus was purified from the ETEC E9034A∆lngA strain [20] using a ZymoPURE™ II Plasmid Maxiprep Kit (Zymo Research, Irvine, CA, USA). The lngA gene was previously replaced with a kanamycin cassette using the one-step inactivation method [23]. Mobilization of the plasmid into ECBL was performed via electroporation (BMC Harvard Apparatus, Cambridge, MA, USA) at 1800 V, and the transformed strains were selected by overnight culture on LB agar plates with kanamycin. The positive colonies were named ECBLpE9034A∆lngA (ECBL∆lngA). The DNA product of the ECBL∆lngA strain was confirmed by PCR using specific primers for the lngA, lngD, and lngH genes (Table 3) and by sequencing. In addition, electrocompetent cells derived from the ECBL∆lngA strain were transformed by electroporation with 100 ng of the pJET1.2 plasmid, which carried variants of the lngA gene of the clinical ETEC strains selected for this study ( Table 4). The transformed colonies were considered positive when cultured on LB agar plates with both kanamycin and ampicillin.

Site-Directed Mutagenesis of the lngA Gene
In this study, the LngA sequence obtained from the ETEC FMU073332 strain was selected for site-directed mutagenesis. In a previous study, ETEC E9034A, FMU073332, and strains with isogenic mutations in the lngA gene were compared according to their adhesion ability. ETEC FMU073332 was more adherent than E9034A [12]; and complete genome data are available for ETEC FMU073332 (NZ_CP017844.1). To identify the variability in the amino acid residues, the FMU07332 LngA variant was cloned into the pJET 2.0 vector to generate pJETlngA FMU073332 , and following the QuikChange protocol provided by Stratagene, the vector was transformed into ECBL∆lngA to restore the lngA gene with specific mutations.
Point substitutions of the amino acid residues in the sequence of the LngAFMU073332 protein based on the LngA E9034A protein were performed according to terminal carboxyl variability, as presented in detail in Table 5. All constructs were verified by DNA sequencing. ECBL∆lngAp T-Q Glutamine (Q), threonine (T), alanine (A), glycine (G), asparagine (N), serine (S), aspartate (D), lysine (K), glutamic acid (E), and arginine (R).

Genome Sequencing
The genomic DNA of the ECBL∆lngA/p FMU073332 strain was extracted using a genomic DNA purification kit (Thermo Fisher, NY, USA). The DNA was used to construct an Oxford Nanopore library following the Genomic DNA by Ligation protocol (SQK-LSK109) (Oxford Nanopore Technologies, ONT). The DNA was not sheared but rather used directly for subsequent steps ranging from purification to library construction. Reads were obtained with the MinION ONT device using the MinION R9.4.1 flow cell. In total, 164,952 reads were obtained. Base calling was performed using Guppy software (v4.0.14), and adapter sequence removal was performed with Porechop (v0.2.4; https://github.com/rrwick/ Porechop accessed on 8 February 2023). The raw reads were aligned against an in silico reconstruction of the ECBL∆lngA/pJETlngA FMU073332 (ECBL∆lngA/p FMU073332 ) genome with bowtie2 (2.3.4.1) [24]. The read mappings were visualized using the Artemis genome visualization tool [25].

Reverse Transcription Polymerase Chain Reaction (RT-PCR)
Total RNA was extracted after all strains (wild type, recombinant ECBL strains with lngA variants, and recombinant ECBL strains with site-specific lngA mutations) were grown overnight in LB medium using an RNeasy Mini Kit (QIAGEN, Hilden, NRW, Germany) and treated with DNase I according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). RT-PCR was performed using the OneStep RT-PCR Kit (QIAGEN, Hilden, NRW, Germany). The RT-PCR conditions were as follows: one cycle of 60 • C for 30 min, one cycle of 95 • C for 15 min, 30 cycles of 95 • C for 1 min, 62 • C for 1 min, and 72 • C for 1 min, and one final extension at 72 • C for 10 min. The RT-PCR products were electrophoresed in 1.5% agarose gels (Promega, Madison, WI, USA) using 1% TAE buffer (Tris-acetate-EDTA). The specific primers used in the RT-PCR assays are described in Table 3. The 16S gene was used as an internal control.

SDS-PAGE and Western Blot Analyses
The bacterial strains were resuspended in 1× Laemmli buffer, boiled for 5 min, and subjected to 16% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) [26]. The separated proteins contained in the acrylamide gels were transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked for 1 h with 5% skim milk in phosphate-buffered saline (PBS), pH 7.4, with 0.5% Tween-20, and the blocked membranes were incubated for 1 h with anti-CS21 polyclonal rabbit or anti-DnaK monoclonal antibodies (MBL International, Woburn, MA, USA) as a loading control. The membranes were washed 3 times with PBS-Tween 0.5% and incubated for 1 h with goat anti-rabbit IgG conjugated to horseradish peroxidase (Sigma-Aldrich Co., St. Luis, MO, USA). The membranes were washed 5 times with 0.5% PBS-Tween and revealed by chemiluminescence-ECL (Amersham Life Science; Arlington Heights, IL, USA). All strains were included in this assay (wild type, recombinant ECBL strains with lngA variants, and recombinant ECBL strains with site-specific lngA mutations).

Statistical Analysis
The data generated from the quantitative analysis of the adherence assays were analyzed using unpaired Student's t-test. In all analyses, a p value less than or equal to 0.05 was considered to indicate statistical significance.

Sequencing of the lngA Gene from Different Clinical ETEC Strains
The lngA gene with its promoter site was amplified from a collection of 12 clinical ETEC strains. The PCR products were purified and sequenced, the nucleotide sequences were translated (https://www.ebi.ac.uk/Tools/st/ accessed on 1 September 2022), and the 12 amino acid sequences without promoters were aligned (http://multalin.toulouse.inra. fr/multalin/ accessed on 5 September 2022) (Figure 1). The lngA promoter regions were also analyzed, and the RNA polymerase-binding sites were determined to be at positions −10 and −35. The alignment showed five nucleotide variations, and no other changes were

Identification of Variable Regions in the LngA Sequences from Clinical ETEC Strains
The amino acid sequences of the  (Figure 1). The amino acid residues at positions 178 to 238 located in the C-terminus contain the main variable regions (Figure 1). In accordance with the amino acid variability in the LngA proteins, three groups were generated: clinical ETEC strains belonging to group V1 (E9034A, 48342, 62123, 63880, and 64760), group V2 (FMU073332, 45162, 49247, 63280, 63283, and 45163), and group V3 (44166) (Figure 1).

Transcription of Recombinant ECBL Strains with lngA Variants, Site-Specific lngA Mutations and Expression of the lngA Gene
The ECBL strain carrying a plasmid that harbored the lng operon with the replacement of the lngA gene with a kanamycin cassette (ECBLpE9034AΔlngA) was complemented with the pJET1.2 vector carrying the lngA variant plus 352 bp upstream of its ATG start codon, including the promoter region of the clinical ETEC strains. Six recombinant ECBL strains with lngA variants were obtained: ECBLΔlngApE9034A, ECBLΔlngApFMU073332,  (Figure 1). The amino acid residues at positions 178 to 238 located in the C-terminus contain the main variable regions (Figure 1). In accordance with the amino acid variability in the LngA proteins, three groups were generated: clinical ETEC strains belonging to group V1 (E9034A, 48342, 62123, 63880, and 64760), group V2 (FMU073332, 45162, 49247, 63280, 63283, and 45163), and group V3 (44166) (Figure 1).

Transcription of Recombinant ECBL Strains with lngA Variants, Site-Specific lngA Mutations and Expression of the lngA Gene
The ECBL strain carrying a plasmid that harbored the lng operon with the replacement of the lngA gene with a kanamycin cassette (ECBLpE9034A∆lngA) was complemented with the pJET1.2 vector carrying the lngA variant plus 352 bp upstream of its ATG  (Table 4). Amplification of the lngA transcript yielded a 609-bp product, and a 22-kDa protein, which corresponded to the LngA protein, was confirmed by Western blot (WB) assays after incubation with anti-CS21 (Figures S2 and S3).
Site-specific mutations in the lngA sequence of FMU073332 were generated according to the C-terminal variation after alignment analysis (some amino acids were replaced by those found in ETEC E9034A) to determine whether these changes affected the ability to adhere to intestinal cells (Figure 2). ECBLpE9034A∆lngA was transformed with pJETlngA FMU073332 vector variants to generate eight recombinant ECBL strains with site-specific lngA mutations: ECBL∆lngAp QTA-TAT , ECBL∆lngAp GN-TT , ECBL∆lngAp T-S , ECBL∆lngAp G-S , ECBL∆lngAp TD-NN , ECBL∆lngAp K-T , ECBL∆lngAp ER-DK , and ECBL∆lngAp T-Q (Table 5). For recombinant ECBL strains with lngA variants, transcription and expression were confirmed by RT-PCR and WB assays (Figures S2 and S3). corresponded to the LngA protein, was confirmed by Western blot (WB) assays after incubation with anti-CS21 ( Figures S2 and S3).
Site-specific mutations in the lngA sequence of FMU073332 were generated according to the C-terminal variation after alignment analysis (some amino acids were replaced by those found in ETEC E9034A) to determine whether these changes affected the ability to adhere to intestinal cells (Figure 2). ECBLpE9034AΔlngA was transformed with pJETlngAFMU073332 vector variants to generate eight recombinant ECBL strains with sitespecific lngA mutations: ECBLΔlngApQTA-TAT, ECBLΔlngApGN-TT, ECBLΔlngApT-S, EC-BLΔlngApG-S, ECBLΔlngApTD-NN, ECBLΔlngApK-T, ECBLΔlngApER-DK, and ECBLΔlngApT-Q (Table 5). For recombinant ECBL strains with lngA variants, transcription and expression were confirmed by RT-PCR and WB assays (Figures S2 and S3).

Adherence to HT-29 Intestinal Cells Was Restored in Recombinant ECBL Strains with lngA Variants
Clinical ETEC strains and recombinant ECBL strains with lngA variants did not show differences in adherence values, independent of the group assignment based on amino acid sequences. ECBLΔlngA was less adherent than E9034A and FMU073332 but showed stronger adherence than the ECBL strain. Almost all of the recombinant ECBL strains with lngA variants showed restored adherence, except two strains that exhibited an increase in adherence compared with the ETEC strains (ECBLΔlngAp48342, ECBLΔlngAp63280 vs. ETEC 48342 and ETEC 63280) (Figure 3). In addition, mannose inhibition assays were performed to demonstrate that adherence was mediated mainly by LngA and not by type 1 fimbriae. The results showed a decrease in adherence with 1% mannose (p = 0.00048; Figure S4), compared with the results obtained from assays without mannose. Our data indicate the existence of other potential adhesins in the ECBL strain, such as the type 1 fimbriae involved in adherence to HT-29 intestinal cells. The qualitative analysis by light microscopy showed a correlation with data generated by the quantitative analysis (Figure 4).

Adherence to HT-29 Intestinal Cells Was Restored in Recombinant ECBL Strains with lngA Variants
Clinical ETEC strains and recombinant ECBL strains with lngA variants did not show differences in adherence values, independent of the group assignment based on amino acid sequences. ECBL∆lngA was less adherent than E9034A and FMU073332 but showed stronger adherence than the ECBL strain. Almost all of the recombinant ECBL strains with lngA variants showed restored adherence, except two strains that exhibited an increase in adherence compared with the ETEC strains (ECBL∆lngAp 48342, ECBL∆lngAp 63280 vs. ETEC 48342 and ETEC 63280) (Figure 3). In addition, mannose inhibition assays were performed to demonstrate that adherence was mediated mainly by LngA and not by type 1 fimbriae. The results showed a decrease in adherence with 1% mannose (p = 0.00048; Figure S4), compared with the results obtained from assays without mannose. Our data indicate the existence of other potential adhesins in the ECBL strain, such as the type 1 fimbriae involved in adherence to HT-29 intestinal cells. The qualitative analysis by light microscopy showed a correlation with data generated by the quantitative analysis (Figure 4).

Recombinant ECBL Strains with Site-Specific Mutations Showed Modified Adherence to HT-29 Intestinal Cells
CS21 pili are oligomers composed of thousands of copies of the LngA protein; we hypothesized that specific amino acids play an important role in the levels of adherence of ETEC strains to HT-29 intestinal cells. Specific substitution of the amino acid residues in the C-terminus of the LngA protein of the FMU073332 strain to amino acid residues in the LngA protein from the E9034A strain was performed to determine their effects on adherence to HT-29 intestinal cells. A decrease in adherence was observed only in four strains with site-specific mutations. This decrease was more evident in the ECBL∆lngAp T-Q strain harboring the substitution of T by Q, which decreased adherence to 86.35%. Analysis of the other strains revealed that reductions were observed with the following substitutions: GN by TT (36.42%), G by S (59.52%), and TD by NN (37.5%) ( Figure 5). The qualitative analysis confirmed that the adherence among the clinical ETEC and recombinant ECBL strains with site-specific lngA mutations was the same (Figure 6). Pathogens 2023, 12, x FOR PEER REVIEW 11 of 18

Recombinant ECBL Strains with Site-Specific Mutations Showed Modified Adherence to HT-29 Intestinal Cells
CS21 pili are oligomers composed of thousands of copies of the LngA protein; we hypothesized that specific amino acids play an important role in the levels of adherence of ETEC strains to HT-29 intestinal cells. Specific substitution of the amino acid residues in the C-terminus of the LngA protein of the FMU073332 strain to amino acid residues in the LngA protein from the E9034A strain was performed to determine their effects on adherence to HT-29 intestinal cells. A decrease in adherence was observed only in four strains with site-specific mutations. This decrease was more evident in the ECBLlngApT- qualitative analysis confirmed that the adherence among the clinical ETEC and recombinant ECBL strains with site-specific lngA mutations was the same (Figure 6).  ECBL∆lngAp FMU073332 was sequenced by Nanopore technology, and a 14-kb plasmid was found to contain the lng operon. The lngA gene was deleted and replaced in this operon by a kanamycin resistance cassette. Moreover, the sequence of pJET1.2 harboring the lngA gene from FMU073332 was identified (PRJNA774369, link: https://www.ncbi.nlm.nih. gov/sra/PRJNA774369 accessed on 8 February 2023), and these data are consistent with the PCR results ( Figure S5). Pathogens 2023, 12, x FOR PEER REVIEW 13 of 18 ECBLΔlngApFMU073332 was sequenced by Nanopore technology, and a 14-kb plasmid was found to contain the lng operon. The lngA gene was deleted and replaced in this operon by a kanamycin resistance cassette. Moreover, the sequence of pJET1.2 harboring the lngA gene from FMU073332 was identified (PRJNA774369, link:

Discussion
The CS21 pilus from ETEC has been described as an adhesin that mediates the interaction among ETEC strains and intestinal cells and induces the formation of bacterial aggregates that protect them from antimicrobial compounds [11,12,27]. A collection of CS21-producing clinical ETEC strains from Mexican and Bangladeshi children with diar-rhea were described as low, moderate, and strong adherents. The ETEC FMU073332 strain exhibits stronger adherence to HT-29 cells and shares its classic virulence factors [toxins (LT and ST) and CFs (CS3 and CS21)] with E9034A, as described by genome analysis [12,20].
In this study, the different adherence levels of clinical ETEC strains to HT-29 intestinal cells, as previously characterized, could be attributed to a repertoire of several CFs, including the CS21 pilus [12]. The lngA gene from 12 strains was selected and sequenced for this assessment. The lngA nucleotide sequence was translated to an amino acid sequence (234 to 236). Other studies have revealed that the sequences of the lngA gene of clinical ETEC strains have a length of 621 nucleotides, translated to 206 amino acid residues [15,17]. The difference in length between the LngA protein sequences is due to the inclusion of a signal peptide of 30 amino acid residues at the N-terminal end, and the prepellin peptidase removes this peptide to generate a mature protein [28]. Because each pilus filament is composed of thousands of pilin subunits, small differences in surface-exposed residues can significantly impact the affinity to its receptor on intestinal cells [29]. An analysis of different sequences of the PilA protein, which is the structural subunit of the type IV pilus of Neisseria meningitidis and Neisseria gonorrhoeae, has shown that this protein is divided into three regions: a conserved region (N-terminal), a semivariable region (middle part of the sequence), and a hypervariable region (C-terminal) [30]. We also found similar results for the amino acid sequences of the LngA proteins, probably because two regions were well defined (78 to 108 and 109 and 114), and the C-terminal region was the main variable region. According to 3D structure models of CofA of the CS8 pilus, the corresponding region from 189 to 236 of LngA has an exposed surface, which can participate specifically in interactions with some cellular ligands that have not been described [18,20].
This variability maintains a correlation with the pilin of the other type IV pili [28]. Gomez-Duarte et al. [17] classified the lngA genes into three distinct phylogenetic groups with 103 to 145 mutational events. Group V1 and V2 allelic variants were identified in this study, but another research group demonstrated that the antigenic diversity of lngA indicates significant structural conservation between the group variants [17]. According to these data, we selected six representative strains from the three groups of trees that exhibited different levels of adherence to clone the lngA sequence into ECBL∆lngA-generated recombinant strains with lngA variants. The challenge was to demonstrate whether the CS21 pilus could be assembled in strains different from ETEC and whether their ability to adhere was maintained or modified.
The presence of specific amino acid variations in the LngA protein could modify the stability or affinity of the CS21 pilus based on different levels of ETEC adherence to HT-29 cells. However, only two recombinant ECBL strains with lngA variants displayed an increase in adherence compared with the ETEC strain, and both of these strains belonged to the same subgroup. Clinical ETEC strains from patients with diarrhea in Mexico and Bangladesh have shown different levels of adherence, and these differences are potentially associated with LngA protein variability, as discussed previously [12]. Recent studies have shown that the LngA protein, which assembles into CS21 pili, is highly immunogenic and may inhibit ETEC intestinal shedding [31]. The differences in the adherence percentages of the clinical ETEC strains and their position in the phylogenetic tree could also be attributed to the expression of other adhesins that promote adherence to HT-29 intestinal cells, such as type 1 fimbriae. In other bacteria, replacement of the bfpA gene (encoding the BfpA structural subunit of the BFP pilus of EPEC) by the tcpA gene (encoding the TcpA structural subunit of the TCP pilus) resulted in the production of an immature TcpA protein without complete processing by the prepilin peptidase BfpB; similarly, the bacteria were unable to assemble and produce the TCP pilus, which suggested that the two assembly mechanisms of the BFP and TCP pili are not compatible [32].
In Neisseria meningitidis, natural antigenic variation in the pilin PilE results in the bacterial strains showing differences in their aggregation ability [33]. ECBL carrying the lng operon could express the LngA protein and exhibited assembly of the CS21 pilus. The presence of variable regions in proteins with adhesion properties, such as the LngA protein, confers adaptive advantages in bacterial pathogenesis, including immune system evasion [29]. Other studies have shown that a variable region of 12 amino acids in the N-terminus of the BabA protein in Helicobacter pylori promotes different levels of adherence [34]. The M protein is the major virulence determinant of Streptococcus pyogenes and has a hypervariable region that gives it the ability to evade cellular phagocytosis [35,36].
Antigenic variations were observed at the predicted LngA surface accessible to Bcell epitopes, and the number of B-cell epitopes was sufficiently high to allow antigen recognition among different LngA variants [31]. To demonstrate whether variability in the C-terminus is related to the adherence of two ETEC (E9034A and FMU073332) strains previously described as strongly adherent, ETEC FMU07332 exhibited the highest adherence, and point mutations in which eight amino acids of the ETEC FMU07332 lngA gene were replaced by the E9034A lngA gene sequence were generated, cloned and transformed into ECBL∆lngA. The substitutions of the amino acid residues GN by TT, G by S, TD by NN, and T by Q induced an important and significant reduction in adherence percentages compared with those in the E9034A and FMU73332 strains.
Our data indicate that the arrangement of amino acid residues such as T, K, G, and N could directly modify the interaction with possible ligands of their host, i.e., HT29 intestinal cells. In Borrelia hermsii, a high rate of amino acid variation in the VMP membrane protein is associated with evasion of the host immune system [37]. The accumulation of point mutations can be an adaptive functional tool used by different clinical ETEC strains to alter adhesion properties, as described with the BFP pilus of EPEC BFP and type 1 fimbriae from Salmonella enterica serovar Typhimurium [38].
The presence of amino acid residues with high variability at the C-terminus could be an essential strategy during the structural organization of the LngA protein that efficiently contributes to the correct assembly of the CS21 pilus; however, additional studies are needed.
The variability in the LngA proteins of clinical ETEC strains confers different attributes to the bacteria to promote colonization of intestinal cells and could confer different features to the bacteria to favor interaction with specific receptors located on the surface of HT-29 intestinal cells. These data indicate that CS21 has the potential to be considered a therapeutic target in the production of a potential vaccine. In conclusion, the LngA protein of clinical ETEC strains gives ECBL the ability to adhere to HT-29 intestinal cells, and the variability and point mutations in this protein modified their adhesion ability. These findings confirm the role of the CS21 pilus in the bacterial adherence process.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/pathogens12020337/s1. Figure S1. Alignment of the lngA nucleotide sequence promoter region. Figure S2. RT-PCR assays for lngA gene detection. Figure S3. Immunodetection by Western blotting of clinical ETEC strains (E9034A and FMU073332), ECBL (E.coli BL21), recombinant ECBL strains with lngA variants and sitespecific mutation in lngA strains. Figure S4. Quantitative analysis of bacterial adherence to HT-29 cells. Figure S5. PCR asayas for the lngD, lngH and lngA. Data Availability Statement: The datasets presented in this manuscript can be found in online repositories. The names of the repositories/repositories and accession number(s) can be found at the following link: https://github.com/ariadnnacruz/CS21-LngA.git (accessed on 5 December 2022).